Emerging technologies for food and drug safety.

Emerging technologies are playing a major role in the generation of new approaches to assess the safety of both foods and drugs. However, the integration of emerging technologies in the regulatory decision-making process requires rigorous assessment and consensus amongst international partners and research communities. To that end, the Global Coalition for Regulatory Science Research (GCRSR) in partnership with the Brazilian Health Surveillance Agency (ANVISA) hosted the seventh Global Summit on Regulatory Science (GSRS17) in Brasilia, Brazil on September 18-20, 2017 to discuss the role of new approaches in regulatory science with a specific emphasis on applications in food and medical product safety. The global regulatory landscape concerning the application of new technologies was assessed in several countries worldwide. Challenges and issues were discussed in the context of developing an international consensus for objective criteria in the development, application and review of emerging technologies. The need for advanced approaches to allow for faster, less expensive and more predictive methodologies was elaborated. In addition, the strengths and weaknesses of each new approach was discussed. And finally, the need for standards and reproducible approaches was reviewed to enhance the application of the emerging technologies to improve food and drug safety. The overarching goal of GSRS17 was to provide a venue where regulators and researchers meet to develop collaborations addressing the most pressing scientific challenges and facilitate the adoption of novel technical innovations to advance the field of regulatory science.

Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells.

Many usnic acid-containing dietary supplements have been marketed as weight loss agents, although severe hepatotoxicity and acute liver failure have been associated with their overuse. Our previous mechanistic studies revealed that autophagy, disturbance of calcium homeostasis, and ER stress are involved in usnic acid-induced toxicity. In this study, we investigated the role of oxidative stress and the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells. We found that a 24-h treatment with usnic acid caused DNA damage and S-phase cell cycle arrest in a concentration-dependent manner. Usnic acid also triggered oxidative stress as demonstrated by increased reactive oxygen species generation and glutathione depletion. Short-term treatment (6 h) with usnic acid significantly increased the protein level for Nrf2 (nuclear factor erythroid 2-related factor 2), promoted Nrf2 translocation to the nucleus, up-regulated antioxidant response element (ARE)-luciferase reporter activity, and induced the expression of Nrf2-regulated targets, including glutathione reductase, glutathione S-transferase, and NAD(P)H quinone oxidoreductase-1 (NQO1). Furthermore, knockdown of Nrf2 with shRNA potentiated usnic acid-induced DNA damage and cytotoxicity. Taken together, our results show that usnic acid causes cell cycle dysregulation, DNA damage, and oxidative stress and that the Nrf2 signaling pathway is activated in usnic acid-induced cytotoxicity.

Overcoming chemoresistance in prostate cancer with Chinese medicine Tripterygium wilfordii via multiple mechanisms.

A leading cause of cancer chemotherapy failure is chemoresistance, which often involves multiple mechanisms. Chinese medicines (CM) usually contain multiple components which could potentially target many mechanisms simultaneously and may offer an advantage over single compounds that target one mechanism at a time. The purpose of this study was to investigate the chemosensitizing effect (CE) of a specific CM, Tripterygium wilfordii (TW), on prostate cancer cells resistant to docetaxel (Dtx) and identify the potential mechanisms. The CE of TW (in combination with Dtx) was evaluated in two Dtx resistant prostate cancer cell lines (PC3-TxR and DU145-TxR) and the efficacy of the combination for resistant PC3-TxR tumor was investigated using a xenograft mouse model. For mechanistic study, the inhibitory effect of TW on P-glycoprotein activity was assessed. In addition, novel gene targets of TW were identified using DNA microarray and quantitative PCR. Results showed that TW induced a CE of 8 and >38 folds in PC3-TxR and DU145-TxR cells, respectively with Dtx IC50 reversed back to that of the sensitive parent cells. An optimum dose of TW+Dtx significantly retarded tumor growth in mice compared to TW or Dtx alone. TW inhibited P-glycoprotein activity and induced a significant gene expression changes in genes related to angiogenesis, cell cycle regulation and differentiation. Our in vitro and in vivo studies demonstrate that TW in combination with Dtx was able to overcome the chemoresistance and suppress resistant prostate tumor growth via multi-mechanisms.

A testing strategy to predict risk for drug-induced liver injury in humans using high-content screen assays and the 'rule-of-two' model.

Drug-induced liver injury (DILI) is a major cause of drug failures in both the preclinical and clinical phase. Consequently, improving prediction of DILI at an early stage of drug discovery will reduce the potential failures in the subsequent drug development program. In this regard, high-content screening (HCS) assays are considered as a promising strategy for the study of DILI; however, the predictive performance of HCS assays is frequently insufficient. In the present study, a new testing strategy was developed to improve DILI prediction by employing in vitro assays that was combined with the RO2 model (i.e., 'rule-of-two' defined by daily dose ≥100 mg/day & logP ≥3). The RO2 model was derived from the observation that high daily doses and lipophilicity of an oral medication were associated with significant DILI risk in humans. In the developed testing strategy, the RO2 model was used for the rational selection of candidates for HCS assays, and only the negatives predicted by the RO2 model were further investigated by HCS. Subsequently, the effects of drug treatment on cell loss, nuclear size, DNA damage/fragmentation, apoptosis, lysosomal mass, mitochondrial membrane potential, and steatosis were studied in cultures of primary rat hepatocytes. Using a set of 70 drugs with clear evidence of clinically relevant DILI, the testing strategy improved the accuracies by 10 % and reduced the number of drugs requiring experimental assessment by approximately 20 %, as compared to the HCS assay alone. Moreover, the testing strategy was further validated by including published data (Cosgrove et al. in Toxicol Appl Pharmacol 237:317-330, 2009) on drug-cytokine-induced hepatotoxicity, which improved the accuracies by 7 %. Taken collectively, the proposed testing strategy can significantly improve the prediction of in vitro assays for detecting DILI liability in an early drug discovery phase.

Utilization of gene expression signature for quality control of traditional Chinese medicine formula Si-Wu-Tang.

The current study utilized a combined pharmacokinetic and genomic approach to demonstrate the feasibility of a new quality control method by using a panel of special differentially expressed genes (DEGs) as unique fingerprint to serve as marker of in vivo bioactivity for a representative traditional Chinese medicine (TCM) formula, Si-Wu-Tang (SWT). The method involves firstly obtaining possible in vivo active components, i.e., the "absorbable" components from the permeate of the Caco-2 monolayer model to simulate oral administration of two specific SWT products (CU-SWT, J-SWT), their component single herbs (Angelicae, Chuanxiong, Paeoniae, and Rehmanniae), and a standard mixture of active compounds (ferulic acid, ligustilide, senkyunolide A). Then, these respective absorbable components were incubated with MCF-7 cells to determine the gene expression profile using microarray processing/analysis as well as real-time PCR. From the available DEGs identified following the incubation, the magnitude of change in DEGs by real-time PCR was found to be consistent with that by microarray. The designated DEGs from the CU-SWT permeate were found to be distinct from other 19 products. Furthermore, the changes in the DEGs resulting from MCF-7 cells treated by eight replicate extracts of CU-SWT on three separate days were consistent. These results demonstrated sufficient specificity and consistency of the DEG panel which could serve as a unique bioactive "fingerprint" for the designated SWT product. The present method for DEG determination may be applied to other TCM products and with further definitive study can potentially provide a unique method for quality control of TCM in the future.

Transcriptional profiling of Chinese medicinal formula Si-Wu-Tang on breast cancer cells reveals phytoestrogenic activity.

BACKGROUND: Si-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women's diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of ß-estradiol (E2) on MCF-7 cells in the Connectivity Map database. METHODS: Further data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells. RESULTS: Many estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT's estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells. CONCLUSIONS: These results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases and possibly for cancer prevention. This study also demonstrated the feasibility of using microarray transcriptional profiling to discover phytoestrogenic components that are present in natural products.

Methysticin and 7,8-dihydromethysticin are two major kavalactones in kava extract to induce CYP1A1.

Kava is a plant traditionally used for making beverages in Pacific Basin countries and has been used for the treatment of nervous disorders in the United States. The pharmacological activity of kava is achieved through kavalactones in kava extract, which include kawain, 7,8-dihydrokawain, yangonin, 5,6-dehydrokawain, methysticin, and 7,8-dihydromethysticin. Recent studies have shown that kava extract induces hepatic CYP1A1 enzyme; however, the mechanisms of CYP1A1 induction have not been elucidated, and the kavalactones responsible for CYP1A1 induction have not yet been identified. Using a combination of biochemical assays and molecular docking tools, we determined the functions of kava extract and kavalactones and delineated the underlying mechanisms involved in CYP1A1 induction. The results showed that kava extract displayed a concentration-dependent effect on CYP1A1 induction. Among the six major kavalactones, methysticin triggered the most profound inducing effect on CYP1A1 followed by 7,8-dihydromethysticin. The other four kavalactones (yangonin, 5,6-dehydrokawain, kawain, and 7,8-dihydrokawain) did not show significant effects on CYP1A1. Consistent with the experimental results, in silico molecular docking studies based on the aryl hydrocarbon receptor (AhR)-ligand binding domain homology model also revealed favorable binding to AhR for methysticin and 7,8-dihydromethysticin compared with the remaining kavalactones. Additionally, results from a luciferase gene reporter assay suggested that kava extract, methysticin, and 7,8-dihydromethysticin were able to activate the AhR signaling pathway. Moreover, kava extract-, methysticin-, and 7,8-dihydromethysticin-mediated CYP1A1 induction was blocked by an AhR antagonist and abolished in AhR-deficient cells. These findings suggest that kava extract induces the expression of CYP1A1 via an AhR-dependent mechanism and that methysticin and 7,8-dihydromethysticin contribute to CYP1A1 induction. The induction of CYP1A1 indicates a potential interaction between kava or kavalactones and CYP1A1-mediated chemical carcinogenesis.

Discovery of molecular mechanisms of traditional Chinese medicinal formula Si-Wu-Tang using gene expression microarray and connectivity map.

To pursue a systematic approach to discovery of mechanisms of action of traditional Chinese medicine (TCM), we used microarrays, bioinformatics and the "Connectivity Map" (CMAP) to examine TCM-induced changes in gene expression. We demonstrated that this approach can be used to elucidate new molecular targets using a model TCM herbal formula Si-Wu-Tang (SWT) which is widely used for women's health. The human breast cancer MCF-7 cells treated with 0.1 µM estradiol or 2.56 mg/ml of SWT showed dramatic gene expression changes, while no significant change was detected for ferulic acid, a known bioactive compound of SWT. Pathway analysis using differentially expressed genes related to the treatment effect identified that expression of genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) cytoprotective pathway was most significantly affected by SWT, but not by estradiol or ferulic acid. The Nrf2-regulated genes HMOX1, GCLC, GCLM, SLC7A11 and NQO1 were upregulated by SWT in a dose-dependent manner, which was validated by real-time RT-PCR. Consistently, treatment with SWT and its four herbal ingredients resulted in an increased antioxidant response element (ARE)-luciferase reporter activity in MCF-7 and HEK293 cells. Furthermore, the gene expression profile of differentially expressed genes related to SWT treatment was used to compare with those of 1,309 compounds in the CMAP database. The CMAP profiles of estradiol-treated MCF-7 cells showed an excellent match with SWT treatment, consistent with SWT's widely claimed use for women's diseases and indicating a phytoestrogenic effect. The CMAP profiles of chemopreventive agents withaferin A and resveratrol also showed high similarity to the profiles of SWT. This study identified SWT as an Nrf2 activator and phytoestrogen, suggesting its use as a nontoxic chemopreventive agent, and demonstrated the feasibility of combining microarray gene expression profiling with CMAP mining to discover mechanisms of actions and to identify new health benefits of TCMs.

Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes.

In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites.

Analysis of gene expression changes in relation to toxicity and tumorigenesis in the livers of Big Blue transgenic rats fed comfrey (Symphytum officinale).

BACKGROUND: Comfrey is consumed by humans as a vegetable and a tea, and has been used as an herbal medicine for more than 2000 years. Comfrey, however, is hepatotoxic in livestock and humans and carcinogenic in experimental animals. Our previous study suggested that comfrey induces liver tumors by a genotoxic mechanism and that the pyrrolizidine alkaloids in the plant are responsible for mutation induction and tumor initiation in rat liver. RESULTS: In this study, we identified comfrey-induced gene expression profile in the livers of rats. Groups of 6 male transgenic Big Blue rats were fed a basal diet and a diet containing 8% comfrey roots, a dose that resulted in liver tumors in a previous carcinogenicity bioassay. The animals were treated for 12 weeks and sacrificed one day after the final treatment. We used a rat microarray containing 26,857 genes to perform genome-wide gene expression studies. Dietary comfrey resulted in marked changes in liver gene expression, as well as in significant decreases in the body weight and increases in liver mutant frequency. When a two-fold cutoff value and a P-value less than 0.01 were selected, 2,726 genes were identified as differentially expressed in comfrey-fed rats compared to control animals. Among these genes, there were 1,617 genes associated by Ingenuity Pathway Analysis with particular functions, and the differentially expressed genes in comfrey-fed rat livers were involved in metabolism, injury of endothelial cells, and liver injury and abnormalities, including liver fibrosis and cancer development. CONCLUSION: The gene expression profile provides us a better understanding of underlying mechanisms for comfrey-induced hepatic toxicity. Integration of gene expression changes with known pathological changes can be used to formulate a mechanistic scheme for comfrey-induced liver toxicity and tumorigenesis.

Gene expression profiles distinguish the carcinogenic effects of aristolochic acid in target (kidney) and non-target (liver) tissues in rats.

BACKGROUND: Aristolochic acid (AA) is the active component of herbal drugs derived from Aristolochia species that have been used for medicinal purposes since antiquity. AA, however, induced nephropathy and urothelial cancer in people and malignant tumors in the kidney and urinary tract of rodents. Although AA is bioactivated in both kidney and liver, it only induces tumors in kidney. To evaluate whether microarray analysis can be used for distinguishing the tissue-specific carcinogenicity of AA, we examined gene expression profiles in kidney and liver of rats treated with carcinogenic doses of AA. RESULTS: Microarray analysis was performed using the Rat Genome Survey Microarray and data analysis was carried out within ArrayTrack software. Principal components analysis and hierarchical cluster analysis of the expression profiles showed that samples were grouped together according to the tissues and treatments. The gene expression profiles were significantly altered by AA treatment in both kidney and liver (p < 0.01; fold change > 1.5). Functional analysis with Ingenuity Pathways Analysis showed that there were many more significantly altered genes involved in cancer-related pathways in kidney than in liver. Also, analysis with Gene Ontology for Functional Analysis (GOFFA) software indicated that the biological processes related to defense response apoptosis and immune response were significantly altered by AA exposure in kidney, but not in liver. CONCLUSION: Our results suggest that microarray analysis is a useful tool for detecting AA exposure; that analysis of the gene expression profiles can define the differential responses to toxicity and carcinogenicity of AA from kidney and liver; and that significant alteration of genes associated with defense response, apoptosis and immune response in kidney, but not in liver, may be responsible for the tissue-specific toxicity and carcinogenicity of AA.

Phytoestrogens and mycoestrogens bind to the rat uterine estrogen receptor.

Consumption of phytoestrogens and mycoestrogens in food products or as dietary supplements is of interest because of both the potential beneficial and adverse effects of these compounds in estrogen-responsive target tissues. Although the hazards of exposure to potent estrogens such as diethylstilbestrol in developing male and female reproductive tracts are well characterized, less is known about the effects of weaker estrogens including phytoestrogens. With some exceptions, ligand binding to the estrogen receptor (ER) predicts uterotrophic activity. Using a well-established and rigorously validated ER-ligand binding assay, we assessed the relative binding affinity (RBA) for 46 chemicals from several chemical structure classes of potential phytoestrogens and mycoestrogens. Although none of the test compounds bound to ER with the affinity of the standard, 17beta-estradiol (E(2)), ER binding was found among all classes of chemical structures (flavones, isoflavones, flavanones, coumarins, chalcones and mycoestrogens). Estrogen receptor relative binding affinities were distributed across a wide range (from approximately 43 to 0.00008; E(2) = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.